MAPK (mitogen activated protein kinase) cascades are conserved from yeast to mammals. In the S. cerevisiae mating pathway, the MAPK module consists of the sequentially acting kinases Ste 11, Ste7, and Fus3/Kss1 that physically associate in a complex with the Ste5 scaffolding protein. The necessity for this assembly is unknown but it may facilitate signal transduction and/or signal insulation to prevent cross-talk among the MAPK pathways. Understanding how the associations are regulated and the consequences for changes in the assembly are unknown. These questions are the foundation upon which our two specific aims have been established: (Aim 1) We have proposed a model whereby the MAPK module cycles between productive and nonproductive states. Our hypothesis predicts a phosphatase may regulate this cycle. We will employ a copurification scheme and phosphatase activity assays to test this model. (Aim 2) Also, since intracellular targeting of Fus3 and Ste5 is pheromone inducible, Ste7 may also display different patterns of localization. Furthermore, since Ste7 becomes phosphorylated in the activation loop and in the regulatory domain at feedack sites, the phosphorylation state of Ste7 may influence its localization. We will use GFP-tagged Ste7 variants to analyze localization of Ste7 upon pheromone induction.